Several publications and patent documents are cited throughout the specification in order to describe the state of the art to which this invention pertains. Each of these citations is incorporated herein by reference as though set forth in full.
Blood coagulation FV is a multi-domain protein (A1-A2-B-A3-C1-C2) that circulates as a procofactor with little or no procoagulant activity. Conversion to the active cofactor state (FVa) involves proteolytic removal of a large, heavily glycosylated central B-domain mediated by thrombin (1). FVa reversibly associates with the serine protease factor Xa (FXa) on a negatively charged membrane surface in the presence of calcium ions to form prothrombinase, the physiological activator of prothrombin (2). Since FVa greatly enhances thrombin generation (1), maintaining FV as an inactive procofactor undoubtedly plays a critical regulatory role that has likely evolved to maintain normal hemostasis. Recently, it has been shown that discrete sequences within the B-domain contribute to the mechanism by which FV persists as an inactive procofactor (3, 4). Elimination of this region results in FV activation without the need for proteolytic processing.
FV is widely represented across the vertebrate lineage with high homology found between the A and C domains and poor homology between B-domains (5, 6). Recent genomic data show that while the length of the B-domain is generally conserved among mammals (˜800 residues), it varies considerably in lower vertebrates (i.e. ˜500 residues in Fugu rubripes) (5, 6); the functional implications of these structural changes are not understood. A striking example of major alterations to the B-domain comes from a group of Australian land snakes (Oxyuranus microlepidotus, Pseudonaja textilis, and Oxyuranus scutellatus). These species are unique in that, in addition to circulating FV as part of their hemostatic system, they also have FV in their venom. Venom-derived FV from each of these species shares ˜44% sequence homology with mammalian FV having a similar domain structure (7-9). However, the B-domain length is dramatically shortened compared to human FV (˜46 versus 836 residues). The venom from these snakes is considered the most toxic in the world, is strongly procoagulant and induces, among other symptoms, disseminated intravascular coagulopathy (10). It contains large amounts of FV as well as a FXa-like enzyme, together forming a complex which is a powerful prothrombin activator. This complex from O. scutellatus (oscutarin C) and P. textilis (pseutarin C) has been partially purified and characterized from crude venom (11-15). Both convert prothrombin to thrombin and their activities are enhanced, to varying degrees, by calcium and phospholipid, but not FVa. Furthermore, pseutarin C also appears resistant to activated protein C (APC), potentially contributing to its role as a potent toxin (7).